Exon deletion pattern in duchene muscular dystrophy in north west of iran.

Objective Duchene and Becker Muscular Dystrophy (DMD/ BMD) are x-linked disorders that both are the result of heterogeneous mutations in the dystrophin gene. The frequency and distribution of dystrophin gene deletions in DMD/ BMD patients show different patterns among different populations. This study investigates the deletion rate, type, and distribution of this gene in the Azeri Turk population of North West Iran. Materials &Methods In this study, 110 patients with DMD/ BMD were studied for intragenic deletions in 24 exons and promoter regions of dystrophin genes by using multiplex PCR. Results Deletions were detected in 63 (57.3%) patients, and around 83% localized in the mid-distal hotspot of the gene (on exons 44–52), 21 cases (33.3 %) with single-exon deletions, and 42 cases (66.6%) with multi-exonic deletions. The most frequent deleted exons were exon 50 (15 %) and exon 49 (14%). No deletion was detected in exon 3. Conclusion This study suggests that the frequency and pattern of dystrophin gene deletions in DMD/ BMD in the Azeri Turk population of North West Iran occur in the same pattern when compared with other ethnic groups.


Introduction
Duchenne (OMIM 310200) and its less severe allelic form Becker (OMIM 300376) muscular dystrophy (DMD/ BMD) are common x-linked recessive hereditary neuromuscular diseases. These disorders result from heterogeneous mutations in the dystrophin gene (1)(2). The incidence of DMD is 1 in 3500 and BMD is 1 in 18000 male births (3)(4). The dystrophin gene that spans a distance of more than 2.5 million base pairs is located at the Xp21 locus and is the largest human gene, which consists of 79 exons. Sequencing this huge gene is time-consuming and, fortunately, the deletions in this gene are non-randomly distributed with many of the large gene deletions that occur in the dystrophin gene can be detected in specific hotspot areas of the gene. These hotspots are clustered in two main regions: 1. about 20% at the 5' proximal portion of the gene (exons 1, 3,4,5,8,13,19); and 2. 80% at the mid-distal region i.e. 42-45, 47, 48, 50-53 (5)(6).

Abstract Objective
Duchene and Becker Muscular Dystrophy (DMD/ BMD) are x-linked disorders that both are the result of heterogeneous mutations in the dystrophin gene. The frequency and distribution of dystrophin gene deletions in DMD/ BMD patients show different patterns among different populations. This study investigates the deletion rate, type, and distribution of this gene in the Azeri Turk population of North West Iran.

Materials &Methods
In this study, 110 patients with DMD/ BMD were studied for intragenic deletions in 24 exons and promoter regions of dystrophin genes by using multiplex PCR.
The reading frame rule explains the clinical differences between DMD/ BMD at the molecular level with the deletions that cause shifts in the reading frame of the dystrophin mRNA (out-of-frame) that lead to no or very low production of functional dystrophin resulting in more severe DMD phenotype whereas the milder BMD phenotype occurs if the reading frame is preserved (inframe) and semi functional proteins are produced (7). In about 65% of DMD cases, the mutation of the dystrophin gene are large-scale gene deletions, in approximately 5% are duplications and in 30% are point mutations (8)(9)(10). Due to no effective treatment available for DMD/ BMD at present, an accurate genetic diagnosis may offer prenatal diagnosis for familial DMD/ BMD although about 30% of cases are the result of new mutations (11). The frequency and distribution of dystrophin gene deletions in DMD and BMD patients show that there are different patterns among different populations. In a previous study of authors in East Azerbaijan Province, in 46 male patients with clinical suspicion of DMD/ BMD, 21 (46%) had deletions. This study investigates the rate, types, and distributions of deletions in DMD/ BDM patients (with a relatively large sample) in the Azeri Turk population of North West Iran by using multiplex polymerase chain reaction (MPCR) assays.

Materials & Methods
All patients were evaluated by an expert pediatric neurologist during 2004-2013. Clinical diagnosis in 104 patients was made by using standard clinical diagnosis criteria for DMD/ BMD in conjunction with high serum levels of Creatine Phosphokinase (CPK) at least 10 times the upper limit of normal and compatible electromyographic results (12). In 6 patients, the diagnosis was confirmed by a muscle biopsy. All patients were of Azeri Turk origin (or from North West Iran). Patients (or their parents) were informed about the study and written consent was signed by either the patient or the parents for blood sampling. This study was approved by Tabriz University of Medical Sciences Ethical Committee. Genomic DNA was extracted from peripheral blood leukocytes using standard protocols. For molecular genetic studies, MPCR assays were done using three complementary MPCR assays that detected 26 exons of the dystrophin gene (6). Three separate PCR assays were performed on each patient's DNA sample to amplify 26 dystrophin gene exons (13). The following exons were studied during this protocol: exon number: 3, 4, 6 , 8, 12, 13, 16, 17, 19, 32, 34, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 60; and the promoter zone.

Discussion
Duchenne and Becker muscular dystrophy are common inherited recessive x-linked motor diseases. Mapping and molecular genetic studies have indicated that both are the result of mutations in the huge gene that encodes dystrophin. There are various laboratory techniques to assess deletion of the dystrophin gene. Some studies have used the Southern Blot technique for this purpose, which is difficult and time consuming. Nevertheless, it has applications in identifying deletion and duplication of the whole gene, locating the origin of deletions and duplications to determine the effect of mutation on the reading frame, and, ultimately, to find the exon junctions. A Southern blot permits prognostication of severity by distinguishing in-frame versus frameshift mutations in over 90% of cases. Some studies have However, the population of the Philippines shows that most deletions are at the beginning of the gene. The same results are reported from other countries as well (18)(19)(20). In this respect, a few studies have been conducted in Iran, including a study by Akbari et al. on 100 patients with Duchenne and Becker dystrophy with 52 patients (52%) showed deletion, 81% of deletions were at the end (exons 44-55), and 19% at the first hotspot of gene (exons [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. The majority of deletions were identified in exons 47, 48, and 46 (18)(19)(20)(21). In Galehdari et al. reported that in Ahwaz 17 patients with suspected Duchenne gene deletion was present in 53% of patients, all of which were in exons 44-51 (19)(20)(21)(22). In a previous study, 46 boy patients in East Azerbaijan Province showed that 21 patients (46%) had deletions. The majority of deletions were observed in exons 45, 49, and 50 at a frequency of 14.3%. These exons were present as the most mutationable exons in patients in this province (20)(21)(22)(23). In our study, the rate and distribution of deletions in patients was similar to the deletion pattern in previous studies in Iran and in terms of pattern of deletion and distribution Iran and Turkey are similar (21)(22)(23)(24). The difference in distribution of deletions can be explained by inclusion criteria for diagnosis of cases, for example: biopsy confirmed or clinical suspicion, different methods used for genetic examination i.e. MLPA technique, or cDNA, Southern Blot or multiple PCR or combination of them; and number of exons studied in MPCR. Finally, the variations strongly suggest that sequence differences exist in the introns and that the differences are in agreement with genetic distances among populations. The similarities further suggest that some intronic sequences have been conserved and that those will trigger recurrent deletions (22)(23)(24)(25). In this study, there were 6 patients with Becker, and considering the diversity of the distribution of gene deletion in these patients, and because of the small sample size, statistical analysis was impossible. However, the results of various studies have shown that based on gene deletion patterns, the disease phenotype cannot be determined (23)(24)(25)(26)(27). Among the applied objectives of this study was to enable research groups and patients to participate in new clinical trials through the identification of gene deletion patterns used MPCR, which are cheaper, faster, and easier than Southern Blot; and because of its high speed, it is ideal for prenatal diagnosis. However, it can only allow for the identification of deletions. Therefore, it is advised to concurrently run both tests. Another method is the use of quantitative PCR (QPCR) to determine gene dosage. This method has an important application in determining carriers, but deleted or duplicated exons should first be identified (14)(15)(16). The Multiplex Ligation-dependent Probe Amplification (MLPA) technique allows for the possibility to assess all deletions or duplications of the dystrophin gene. Lai et al. compared MPCR and MLPA and concluded that MLPA detected all known deletions and duplications as well as the extent and distribution of the deletions and duplications, which in turn facilitated a genotypephenotype correlation (17). According to this study, using MPCR in Azeri Turk patients in North West Iran, showed that intragenic deletions were detected in 63 (57.3%) DMD/ BMD patients. There are various reports on the frequency and pattern of intragenic deletions in different populations. According to these reports, in addition to deletion frequency differences in different populations, the distribution of deletions in the dystrophin gene is different. Table 1 presents a summary of rate and distribution patterns of deletion for different countries available in the Leiden Muscular Dystrophy database (http://www.dmd.nl/ DMD_deldup.html). Table 1 shows that in North America and Europe, the deletion rates vary from 42% to 90%, deletion rates in Asian countries vary from 31% to 86%, and the deletion in Latin American countries varied from 47% to 93%. The deletion rate in Africa was reported at 51%. In this study, 83% of deletions were in the mid-distal hotspot of the gene (exons 44-52) and 17% in the 5' proximal portion of the gene (exons [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. The highest frequency of deletion was observed in exon 50 (15%), then followed by exon 49 (14%). 1-B 1-A result in a clinical phenotype like BMD. This AON can be applied to about 13% on the DMD patients. (28)(29)(30).
In conclusion, the present study suggests that the frequency and pattern of dystrophin gene deletions in DMD/ BMD in the Azeri Turk population in North West Iran occur with the same pattern compared with other ethnic groups.

Acknowledgment
This study was supported by the Tabriz University of Medical Sciences. We thank the patients and their parents for participation in this study.